Determining the ability of an enzyme to catalyze the chemical conversion of a substrate to a product is key to understanding a variety of problems in biochemistry and medicine, including the development of novel therapies which alter such reactions. Enzymologists have been adaptable in devising ways to follow an enzymatic reaction in crude systems. For example, in the early development of enzymology, scientists used spectrophotometric analysis of enzymatic reactions where the adsorption spectrum of the product was distinct from the adsorption spectrum of the substrate. Although such early assays had the advantage of continuous monitoring of the enzyme reaction, the change in adsorption at certain points in the spectrum could occur for reasons other than the primary reaction under study, causing unreliable results. Further, in many enzymatic reactions, the product (or substrate) cannot be monitored continuously, and thus the only way to observe the reaction is to use a discontinuous assay where the reaction is allowed to proceed for a set period and then is terminated. Colorimetric assays are an example of a discontinuous assay in which the stopped reaction can be treated in a subsequent procedure that results in a colorimetric reaction that is then quantitated. Colorimetric assays are more reliable than spectophotometric assays, but are limited by their sensitivity.
Even with the increased reliability of the newer enzyme assays, there are many enzymatic reactions in which the separation of reactant and product is necessary before any quantitation can be done. Thus, reliable and sensitive separation assays are needed.
An example of sensitive assays which separate product and substrate include, for example, electropheoresis, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and chromatographic techniques, including thin paper, paper or liquid chromatography, such as high-performance liquid chromatography-, or HPLC-, based separation techniques. Even though these assays allow for reliable quantitation of the enzyme activity, one major disadvantage of these separation techniques is that they are not easily modified for high throughput format, which is useful for the large-scale screening of compound libraries necessary for drug discovery.
Enzyme activity assays have been developed which can be done on 96-well plates, but these too have limitations. In these assays, the reaction is done in the 96 well plate format, and then is stopped by a stop buffer. The resulting product is pored onto and then immobilized or bound to a solid support, such as an insoluble polymeric material, inorganic or organic matrix, or gel, which is then washed repeatedly to remove non-immobilized components. The immobilized components are then quantified. Even though this assay is high-throughput, it is still cumbersome in that it is not done in a single reaction container and requires multiple steps, making automation difficult. Thus, there is a need in the art for a sensitive and reliable enzyme activity assay which stops the reaction and separates the enzyme product from the substrate in a single step, such that it can be used in an automated, high-throughput format.